Mechanisms of a number of human diseases have been elucidated. The recombinant DNA techniques and the techniques for transferring a gene into cells have rapidly progressed. Under these circumstances, protocols for somatic gene therapies for treating severe genetic diseases have been recently developed. More recently, attempts have been made to apply the gene therapy not only to treatment of the genetic diseases but also to treatment of viral infections such as AIDS as well as cancers.
Viral vectors currently used in general include retroviral vectors, adenoviral vectors and adeno-associated viral (AAV) vectors. A retroviral vector can be readily prepared and integrate a foreign gene into the chromosomal DNA of a target cell. Therefore, it is useful for gene therapy for which a long-term gene expression is desired. However, since a foreign gene is integrated at random sites in a chromosomal DNA, cancer or the like may be potentially caused if a retroviral vector is used. Furthermore, since a retroviral vector cannot infect cells in resting phase, the types of target cells are limited.
Although there were problems about the method for preparing adenoviral vectors, a convenient preparation method has been developed. The vector can efficiently infect many types of cells including cells in resting phase. However, since it does not have a mechanism for integrating a foreign gene into the chromosomal DNA of a target cell, the expression of the foreign gene is usually transient.
An AAV vector can infect cells in resting phase and has a nature to integrate a foreign gene specifically at the AAVS1 site on human chromosome 19. Thus, it is expected that a gene transferred using the AAV vector is expressed for a long period in a cell without potential risk. However, there are practical problems associated with the AAV vectors that the preparation of AAV is complicated and the size of a gene that can be transferred into cells is very small.
As described above, conventional viral vectors used for gene therapy have their own advantages and disadvantages. No gene transfer method that enables convenient handling, high gene transfer efficiency and long-term expression of a transferred gene was known. A system that satisfies these properties has been desired.
An attempt was made to develop such a system. In the system, a region of AAV required for the site-specific integration of a foreign gene is incorporated into an adenoviral vector which has advantages of high gene transfer efficiency and easy preparation of a high-titer vector in order to overcome the drawback of the adenoviral vectors that they cannot integrate a foreign gene into the chromosomal DNA of a target cell (see, for example, U.S. Pat. No. 5,843,742).
However, since a Rep protein, which is encoded by the region of AAV required for the site-specific integration of a foreign gene, inhibits the proliferation of an adenovirus, an adenovirus having the region cannot proliferate. Therefore, it is impossible to prepare such a vector and, in consequence, gene transfer using such a vector is practically infeasible.
Recchia et al. tried to solve the above-mentioned problem by using a hepatic cell-specific promoter, α1AT promoter, for expressing a rep gene (Recchia et al., Proceedings of the National Academy of Sciences of the USA, 96:2615–2620 (1999)). Since the α1AT promoter is a hepatic cell-specific promoter, it does not operate in a virus-producer cell to be used for the preparation of an adenoviral vector (a producer cell). Therefore, the rep gene contained in the adenoviral vector is not expressed and the proliferation of the adenoviral vector is not inhibited.
However, the method of Recchia et al. has a drawback that the cell type in which a foreign gene can be integrated into the chromosomal DNA is limited to the hepatic cell because the promoter used for expressing the rep gene is a hepatic cell-specific one.
The prior art has drawbacks as described above. A method which results in high gene transfer efficiency, by which a high-titer vector is readily prepared, which enables the integration of a foreign gene into the chromosomal DNA of a target cell, and which can be used to transfer a foreign gene into a wide variety of cell types has been desired.